Abstract This R61/R33 application proposes initial steps to develop pregnenolone (an endogenous neurosteroid made from cholesterol in the brain and adrenals that is sold as an over-the-counter supplement) as an antidepressant in women. Preclinical literature suggests that a pregnenolone analogue is effective in animal models of depression. Low cerebrospinal fluid levels of pregnenolone are reported in humans with depression, low serum levels in anxiety, and low parietal cortex levels in suicide. We conducted two placebo-controlled pilot studies of pregnenolone (100 mg/d and 500 mg/d) in depressed (primarily bipolar) patients. Both studies found good tolerability and greater improvement in depressive symptoms with pregnenolone than with placebo, with a larger effect size at the higher dose. Women showed a more robust response than men in both pilot studies. In addition, improvement in memory was observed with pregnenolone in women. Reduction in depressive symptoms strongly correlated with increases in serum pregnenolone levels. Given the wide availability of pregnenolone, and promising preclinical and clinical data, particularly in women, we propose to develop pregnenolone as an antidepressant. Several questions about pregnenolone need to be resolved before moving into large-scale clinical trials. The pharmacokinetic properties of pregnenolone are not well characterized, optimum dose and trial length have not been identified, and biosignatures associated with pregnenolone response have not been determined. We will use this R61/R33 grant to advance these objectives. In the R61 phase, adult women with mild to moderate major depressive disorder will receive seven days of pregnenolone (500 mg/d, 800 mg/d or placebo) in a randomized double-blind, crossover design, with a 14-day washout period between each condition. Neuroimaging markers pertinent to both depression and pregnenolone (resting state function connectivity, proton spectroscopy) will be assessed after each condition (biosignature engagement). Serum levels of pregnenolone and downstream neurosteroids will be obtained (bioavailability assessment), and relationships between changes in neurosteroids and biosignatures examined. If the R61 phase identifies a biosignature associated with a significant change at a well-tolerated dose, the grant will progress to the R33 phase which will examine a longer exposure to pregnenolone or placebo in a larger sample. Depressive symptoms will be measured, pregnenolone effects on the biosignature will be replicated, and relationships between biosignature and depressive symptom changes will be quantified in the R33 phase. A pharmacokinetic study will determine the optimum daily dosing strategy. An investigative team with extensive experience with clinical trials, neurosteroids, neuroimaging, and pharmacokinetics, as well as an extensive history of research collaboration, will conduct the studies.